Molecular Chain Elongation Mechanism for n ‐Caproate Biosynthesis by Megasphaera Hexanoica

Abstract The microbial production of medium‐chain carboxylates has attracted considerable interest owing to their potential applications in biofuels and specialty chemicals; however, the underlying biosynthetic mechanisms remain incompletely understood. The present study evaluates the medium‐chain carboxylate‐producing microbe Megaspahera hexanoica using genomic analysis, transcriptome analysis, and metabolic engineering. Additionally, the n ‐caproate synthesis pathway of M. hexanoica is characterized with fructose as an electron donor, and the substrate specificity of the respective proteins is evaluated by constructing an n ‐caproate biosynthetic pathway in Escherichia coli . Among all r‐BOX or RBO genes, thl _1583, which encodes β‐ketothiolase ( Mh THL), is identified as the most critical enzyme for the carbon chain elongation mechanism in M. hexanoica . Therefore, Mh THL is compared with other well‐studied β‐ketothiolases ( Ck THL from Clostridium kluyveri, Re BktB from Ralstonia eutropha ( Cupriavidus necator ), Ec AtoB from E. coli , and Ca THL from C. acetobutylicum ). Mh THL is found to exhibit the highest n ‐caproate production, as evidenced by the protein crystal structure of Mh THL. Structural comparisons with other thiolases show that Mh THL has a larger substrate‐binding pocket than Re BktB. Thiolase mutants generated by site‐directed mutagenesis reveal that two residues (Leu87 and Val351) are essential for determining the size of the substrate‐binding pocket.